Quantitation of Aflatoxins B1, B2, G1, G2 and Ochratoxin a in Cannabis

No. SSI-HPLC-019

High Performance Liquid Chromatography

Quantitation of Aflatoxins B1, B2, G1, G2 and

Ochratoxin A in Cannabis by LC Using Prominence-i and the RF-20Axs Fluorescence No. HPLC-019 Detector

■ Introduction Aflatoxins and ochratoxins are varieties of The mixture was shaken vigorously for 10 minutes mycotoxins produced as secondary metabolites from using the SPEX Geno Grinder, and then centrifuged mold species within the genera Aspergillus. Studies at 3600 rpm for 3 minutes. 2.0 mL of the have found that many mycotoxins, including supernatant was transferred to a new tube Aflatoxins G1, G2, B1, B2, and Ochratoxin A, are containing 8.0 mL of 1x PBS/2% Tween Wash Buffer immunosuppressive, carcinogenic, neurologically and mixed well. All 10 ml of the diluted extract was toxic, and hepatotoxic [1,3]. The mold itself can also passed through the AflaOchra column (see Figure 1) cause diseases such as lung infection and at a rate of 1-2 drops per second until air came aspergillosis [1,3]. Considering the great risk to through the column—it is important that the flow consumer health posed by the presence of rate does not exceed 1-2 drops per second when mycotoxins and the possibility of mold growth in using these columns because doing so could reduce cannabis [1,2], the development of accurate testing recovery. Likewise, the column was washed with 10 methods for mycotoxins in cannabis are of utmost mL of PBS/2% Tween buffer, followed by 10 mL of importance. HPLC grade water. Finally, the column was washed with 2 mL of methanol, 1 mL at a time and As more states adopt policy changes allowing for combined in the same tube. medical or recreational use of marijuana, more regulations regarding testing of cannabis products have been implemented. Existing methods include ELISA, qPCR, and MALDI-ToF-MS; each one has their own advantages and disadvantages in terms of cost, range, preparation time, and accuracy. For instance, ELISA is not accurate and often gives false positive or negative results, and additional expensive instrumental tests need to be conducted to confirm the ELISA results. Moreover, due to the high level of matrix complexity of cannabis products, the mycotoxin testing in cannabis is even more challenging. This study focuses on developing a powerful detection method that allows cannabis testing labs to confidently report concentrations below regulatory limits – Aflatoxins B1, B2, G1 and G2 combined to 20 ppb and 20 ppb for Ochratoxin A alone.

■ Experimental Sample Preparation One gram of sample was weighed out in a 50 ml centrifuge tube and ground using a SPEX grinder until homogenized. 10 mL of methanol: water

(80:20) was then added to the tube. Figure 1: Afla-Ochra Column for extraction/clean-up No. SSI-HPLC-019

Standards and Calibration The standards used to build the calibration curve were ran in triplicate using the high-sensitivity mode at 2, 5, 10, 14, 20, and 50 ppb for the Aflatoxins and 10, 14, 20, and 50 ppb for Ochratoxin A. All standards were spiked in matrix and extracted so the actual concentrations of the injected material were 0.2, 0.5, 1.0, 1.4, 2, and 5ppb for the Aflatoxins. The Ochratoxin A does not need as low of a detection limit because the limit is 20 ppb by itself; the Aflatoxins is 20 ppb for the sum of the four.

Analytical Conditions Column NexLeaf CBX for Potency 2.7 um x 150 mm x 4.6 mm Mobile Phase A: Water with 0.1% Formic Acid, B: Methanol with 0.1% Formic Acid, C: Acetonitrile with 0.1% Formic Acid Time Conc. A/Conc. B/Conc. C = 65/30/5 Figure 2: Chromatography of Aflatoxin G1 2 ppm (left) and Program (0.00 – 2.00) → ramp to 23/40/37 (2.00 Ochratoxin A 10 ppm (right) – 10.00) → 23/40/37 (10.00 – 12.00) → 65/30/5 (12.01 – 14.00) Flow Rate 1.3 mL/min Column 50 °C Temp. Injection 10 uL Volume Detection RF-20AXS, Channel 1: Ex= 365nm EMm=450nm, Ch2: Ex=336, Em=464, Ch3: Ex=330 Em=460, Ch4: Ex=350 Em=450, High Sensitivity Mode Cell Temp. 30 °C

The same column and LC system (i-Series LC-2030) as used here are also used for cannabis potency analysis, so a cannabis lab can expand their analysis capabilities by adding just the RF-20AXS to their current setup.

■ Results and Discussion Channel 1, excitation of 365 nm and emission at 450 nm, provided the best response for all Aflatoxins and Channel 3, excitation of 330 nm and emission at 460 nm, was chosen for Ochratoxin A, though Channel 2 was also suitable (see Figure 2). Aflatoxin G1 had the lowest response of all the Aflatoxins, but Channel 1 still provided quality data for analysis even at the lowest concentration of 2 ppm. Calibration curve R^2 values for each compound were 0.998 or greater, indicating great linearity. Figure 3: Calibration curves for Aflatoxin G1 (top) and Ochratoxin A (bottom)

No. SSI-HPLC-019

Samples were spiked at 10 ppb of mycotoxins and ■ Conclusion run as unknowns. The chromatograms are shown in A new fluorescence method has been developed to Figure 4 and the recovery results, indicating the high test these Mycotoxins in cannabis. This method is accuracy of the method, are shown in Table 1. sensitive and robust and requires only the simple addition of our RF-20AXS detector to the i-Series LC- 2030.

■ References 1) Hazenkamp A. An evaluation of the quality of medicinal grade cannabis in the Netherlands. Cannabinoids. 2006; 1(1):1-9. 2) Llewellyn G. C., O’Rear C. E. Examination of fungal growth and aflatoxin production on marihuana. Mycopathologia. 1977; 62(2): 109-112. 3) Sedmikova M., Reisnerova H., Dufkova Z., Barta I., Jilek F. Potential hazard of simultaneous occurrence of aflatoxin B1 and ochratoxin A. Veterinary Medicine - Czech. 2001; 46(6): 169-174.

Figure 4: Full chromatogram showing Channels 1 (top) and 3 (bottom) for 10 ppb spiked sample

Table 1: 10 ppb spiked sample results

Mycotoxin Concentration Recovery (%) Concentration (ppb) Recovery (%) (ppb) Aflatoxin G2 8.022 80.22 8.380 83.80 Aflatoxin G1 8.534 85.34 8.958 89.58 Aflatoxin B2 7.901 79.01 8.363 83.63 Aflatoxin B1 7.704 77.04 8.044 80.44 Ochratoxin A 8.781 87.81 10.925 109.25

First Edition: April 2018

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